摘要
目的 构建stathmin基因真核表达载体,研究上调stathmin基因表达对食管癌细胞EC9706的作用.方法 采用RT-PCR扩增stathmin cDNA,克隆至pcDNA3.1（＋）真核表达载体.重组载体经脂质体转染EC9706细胞,G418筛选获得稳定转染细胞株.显微镜观察转染细胞形态学变化,Western印迹法检测转染细胞stathmin蛋白的表达.选取稳定转染的细胞株,采用细胞计数法绘制细胞生长曲线,四甲基偶氮唑盐法检测细胞增殖活性,平板克隆形成实验观察细胞克隆形成,流式细胞仪分析细胞周期,裸鼠移植瘤实验研究转染细胞的成瘤性.结果 RT-PCR扩增获得450 bp的cDNA编码序列;克隆至pcDNA3.1（＋）载体,获得pcDNA3.1-stathmin重组表达载体.Western印迹法证实重组载体上调EC9706细胞stathmin蛋白表达,差异有统计学意义（P＜0.01）.与对照组细胞比较,pcDNA3.1-stathmin转染细胞形态变大、多核、有丝分裂障碍;pcDNA3.1-stathmin转染细胞倍增时间延长（25～28 h）、增殖活性降低、细胞克隆形成率下降（34.5%±6.9%）,细胞分裂阻滞于G2/M期（21.7%±3.4%）,裸鼠体内致瘤性降低,差异均有统计学意义（均P＜0.01）.结论 重组质粒pcDNA3.1-stathmin上调stathmin基因表达能够抑制食管鳞癌EC9706细胞的增殖和致瘤性,stathmin可能成为食管鳞癌患者治疗的理想靶点.
Objective To construct a eukaryotic expression vector of stathmin gene and investigate its effect on esophageal squamous cell carcinoma （ ESCC） EC9706 cell line. Methods Stathmin cDNA coding sequence was amplified by RT-PCR and cloned into a eukaryotic expression vector pcDNA3. 1（ ＋ ）. EC9706 cells were transfected with this recombinant plasmid pcDNA3. 1-stathmin by lipofectamine. And the stable transfectants were selected with G418 medium. Stathmin protein expression was detected with Western blot in transfected EC9706 cell lines. Morphologic change of stable transfectants was observed under microscope. The proliferation of transfected cells was measured by cell counting, MTT and in vitro formation assay of flat Flow cytometry was used to detect the cell cycle. Nude mice were adopted to investigate the in vivo tumorigenic characteristics of the transfected cells. Results A 450 bp coding sequence of stathmin cDNA was amplified by RT-PCR and then cloned into pcDNA3. 1 （ ＋ ） plasmid to harvest the recombinant plasmid pcDNA3. 1-stathmin. The recombinant plasmid pcDNA3. 1-stathmin and blank vector were transfected respectively into EC9706 cells. The up-regulated expression of stathmin protein was validated by Western blot （ P 〈 0. 01 ） . Compared with control, EC9706 cells transfected with pcDNA3. 1-stathmin appeared swollen and multi-nuclear with a cell mitotic arrest; doubling generation time of pcDNA3. 1stathmin transfectants was prolonged（25 -28 h）; The in vitro cell proliferation ability and clone formation rate （34.5% ＋-6.9%） decreased, cell cleavage was blocked at G2/M phase （21. 7% ＋- 3. 4% ） and the oncogenicity of inoculated cells in nude mice decreased （ all P 〈 0. 01）. Conclusions The up-regulated expression of stathmin protein triggered by the recombinant plasmid pcDNA3. 1-stathmin can inhibit the proliferation and oncogenicity of ESCC EC9706 cells. TTiis molecule may be a promising therapeutic target in ESCC patients.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2010年第30期2140-2144,共5页
National Medical Journal of China
基金
河南省科技厅科技攻关项目（072102310054）
